New isolates of Trichoderma spp. as biocontrol and plant growth–promoting agents in the pathosystem Pyrenophora teres-barley in Argentina

Journal Pre-proofs Trichoderma as biocontrol and plant growth-promoting agent in the pathosystem Pyrenophora teres-barley in Argentina Moya Paulina, Barrera Viviana, Cipollone Josefina, Bedoya Carolina, Kohan Lucila, Toledo Andrea, N. Sisterna Marina PII: DOI: Reference:

S1049-9644(19)30482-7 https://doi.org/10.1016/j.biocontrol.2019.104152 YBCON 104152

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Biological Control

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30 June 2019 30 October 2019 14 November 2019

Please cite this article as: Paulina, M., Viviana, B., Josefina, C., Carolina, B., Lucila, K., Andrea, T., Sisterna Marina, N., Trichoderma as biocontrol and plant growth-promoting agent in the pathosystem Pyrenophora teres-barley in Argentina, Biological Control (2019), doi: https://doi.org/10.1016/j.biocontrol.2019.104152

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Trichoderma as biocontrol and plant growth-promoting agent in the pathosystem Pyrenophora teres-barley in Argentina Moya Paulina 1,2; Barrera Viviana 4; Cipollone Josefina1,3; Bedoya Carolina1 ; Kohan Lucila1; Toledo Andrea1,2*; Sisterna Marina N. 1,3 1Centro

de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, 60 y 119, 1900, La Plata, Buenos Aires, Argentina. 2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. 3Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICPBA), Argentina. 4Instituto de Microbiología y Zoología Agrícola (IMyZA) Instituto Nacional de Tecnología Agropecuaria. N. Repetto y De los Reseros, C.C. 25 1712, Castelar, Buenos Aires, Argentina. *Corresponding author at: E-mail address: [email protected] Declarations of interest: none

Abstract The fungus Pyrenophora teres (Drechsler), causal agent of net blotch of barley, generates important yield losses, mainly regarding grain weight and quality of malt extract for beer production. In order to find new strategies to manage this disease, we evaluated in vitro and in vivo antagonistic interactions among different native isolates of Trichoderma spp. and P. teres. The microorganisms were isolated from different barley crop areas of Buenos Aires province and identified according to morphological features and molecular techniques. Dual cultures were conducted to assess in vitro antagonism using direct interactions. Assays under greenhouse conditions were carried out to evaluate the efficiency of Trichoderma spp. as biocontrol and plant growth-promoting agent. In vitro, all Trichoderma spp. isolates inhibited mycelial growth of P. teres in a 18%-54% range compared to the controls. Microscopic observations of interaction zones revealed morphological alterations of P. teres such as vacuolated mycelium without typical pigmentation and coiling. In vivo, all Trichoderma isolates significantly decreased the incidence of P. teres, up to 55% on barley seedlings. Severity decreased up to 77% on stems and up to 70% on leaves. Likewise, regarding growth-promoting parameters, aerial and radicular dry weight of plants treated with Trichoderma spp. increased up to 20% and 15%, respectively. On the other hand, an increase of chlorophyll (SPAD) of up to 9% compared to the control was recorded in seedlings treated with Trichoderma. Keywords: Antagonistic effect; Drechslera teres; Hypocrea; net blotch 1. Introduction Net blotch of barley, caused by Pyrenophora teres (Dried.) Drechsler (Ascomycota, Dothidomycetales, Pleosporaceae), is one of the most important diseases affecting this crop in all cerealgrowing regions of the world. Yield losses from 10 to 40% have been recorded in countries such as Australia and Canada (Murray & Brennan 2010; Akhavan et al. 2017). In South America, Argentina is the most important producer of barley for the malting industry, with a reported yield of 4.1 million tons in 2018-2019 (Bolsa de Cereales, 2019). In this country, net blotch of barley causes 20% yield losses and affects the quality of malt for beer production (Carmona et al. 2011). Net blotch lesions on leaves are the main symptom of this disease. The causal agent survives from one season to the next in the stubble from the previous crop and in infected seeds, which are the main source of inoculum (Reis, 1991). Thus, it is necessary to focus research on the health of seeds, due to their role in the spread and dispersion of this pathogen to disease-free areas. Although several studies have shown that net blotch can be successfully controlled using triazoles alone or in combination with strobilurins (Carmona et al. 2011; Akhavan et al. 2017), an indiscriminate use of these substances may lead to the rise of resistant forms of the pathogen and affect both the environment and human health (Baturo-Ciesniewska et al. 2012). Currently, in the context of

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integrated management disease (IMD), biological control is a sustainable alternative to minimize the pollution of natural resources (Boland 1990; Heydari & Pessarakli 2010). The genus Trichoderma Pers. is one of the most widely studied Hypocrealean fungi due to its diverse applications in agriculture, industry, and environmental issues (Mukherjee et al. 2013) The antagonistic properties of Trichoderma species that are used as biocontrol agents are based on several mechanisms. They can also behave as plant growth promoters, inducing increases in weight, root and stem surface and length in crops of economic importance (Perelló & Dal Bello, 2011; Hermosa et al. 2012; Jalali et al. 2017). In Argentina, previous studies using Trichoderma spp. have shown success in the control of pathogens of cereal phylloplanes and seeds (Mónaco et al. 2004; Perelló & Dal Bello 2011; Stocco et al. 2016). The aim of the present work was to assess the effect of native isolates of Trichoderma spp. as biocontrol and plant growth-promoting agent in the P. teres-barley pathosystem, with a previous morphocultural and molecular characterization of the microorganisms. 2. Materials and methods: 2.1 Fungal isolates Eight isolates of Trichoderma spp. and two isolates of P. teres were obtained from barley rhizosphere and seeds, respectively, from four localities in Buenos Aires province, Argentina (namely Tres Arroyos, Bordenave, Bolívar and Barrow). Additionally, one isolate of the pathogen was obtained by phytopathological techniques from symptomatic barley leaves from crops in the Experimental Station Ing. Agr. Julio Hirschhorn, Los Hornos, Buenos Aires province, Argentina. The isolates of Trichoderma spp. were obtained following Elad et al. (1981). P. teres were isolated from seeds applying ISTA methods (Neergaard 1979). All isolates were cultured on potato dextrose agar (PDA Britania) and maintained at 4°C at the collection of Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, until use. 2.2 Morpho-cultural characterization The eight Trichoderma spp. isolates (T0, T2, T3, T4, T7, T8, T9 and T10) were characterized according to their morphology and culture characteristics following the methodology recommended by Samuels et al. (2006 b). For this purpose, 5 mm-diameter mycelial discs were cut from cultures grown on PDA for 7 days at 25 ± 1 °C and 12 h (L:D) photoperiodicity, and transferred to 90 mm Petri dishes containing both Agar Spezieller Nährstoffarmer (SNA) and PDA. Five replicates of each isolate were done and incubated at 25°C and 12 h photoperiod under white fluorescent light. Macroscopic observations including colony morphology, presence of pigment and characteristic odor were made at between 72 and 96 h of incubation. Colony radius was measured and recorded every 24 h (Barrera, 2012), placing an active growth mycelium disc from each isolate on plates with PDA and SNA. Three plates were prepared for each medium and isolate; these were incubated for 96 h at 15, 25 and 35 ° C in darkness according to Jaklitsch (2009). The microscopic observations performed on cultures grown on SNA (Samuels et al. 2006 b) included number of phialides, presence of sterile projections, chlamydospores, and measurements of 40 conidia. The three P. teres isolates (Pt A, Pt C and Pt M) were characterized morphologically following the descriptions of Ellis (1971) and Sivanesan (1987). The colonies were analyzed regarding their coloration, presence of pigments and growth rate after 3 and 6 days of incubation on PDA at 24 C° in darkness. Microscopic observations including record of shape, color and measurements of 40 conidia from each isolate. Microscopic characterization was made under a Nikon YS2 optical microscope equipped with a Nikon D40 digital camera.

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2.3 Molecular characterization

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2.3.1 DNA extraction and phylogenetic analysis of Trichoderma

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The DNA extraction from the eight Trichoderma isolates was performed according to Vazquez et al. (2015). Amplification of the translation elongation factor 1 alpha (tef1) was carried out, given that in groups of complex species such as this, this marker has shown to have enough variability to resolve

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species (Jaklitsch, 2009).The primers Ef728M (5´-CATYGAGAAGTTCGAGAAGG-3´) and Ef2 (5´GGARGTACCAGTSATCATGTT-3´), developed by Samuels et al. (2011) were used. The reaction mixture was made with a final volume of 20 µl containing 5-20 ng of fungal DNA, a mixture of 10 mM of each nucleotide, 0.25 µM of each primer, 1 U of TaqDNA polymerase (Invitrogen, Thermo Fisher Scientific), 1.25 mM of Cl2Mg and 1x reaction buffer (Master mix from New England Biolabs).The program used for the PCR was described by Samuels and Ismiel (2009). Sequencing was made using BigDyeTM Terminator v 3.1 (Applied Biosystems) based on Sanger's method (Sanger & Coulson, 1978). The PCR products were purified using ethanol precipitation and analyzed in a Genetic Analyzer 3130xl at SIGYSA (Argentina). The Trichoderma spp. sequences corresponding to tef1 were edited using the program BioEdit version 7.0.9.0 (Hall, 1999) and used to perform a phylogenetic analysis, which included the sequences from the Trichoderma spp. isolates: T0, T2, T3, T4, T7, T8, T9 and T10, and the sequences of 19 species of the genera Trichoderma / Hypocrea available from the GenBank database (www.ncbi.nlm.nih.gov). Brunneosphaerella jonkershoekensis (Marinc., M.J. Wingf. & Crous) Crous (Ascomycota: Capnodiales) (JN712579) was used as outgroup. The sequences were aligned using the Clustal W Mega5 software (Tamura et al., 2011), assuming a penalty for opening and extending the gap of 15 and 6.66, respectively. The alignment was automatically edited using Gblock software version 0.91b (Talavera & Castresana, 2007), setting the minimum length of a block at 4 and leaving all other options at default values. The phylogenetic study was carried out by maximum likelihood analysis, using 1000 bootstrap replicas to evaluate the statistical support of nodes.

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2.3.2 DNA extraction and phylogenetic analysis of P. teres

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Two P. teres isolates that presented morpho-cultural differences were selected for their molecular characterization, one from seed: Pt M (DtLPS1) and another from diseased plant tissue with the typical symptoms: Pt A (DtLPS2). The Internal transcribed spacer (ITS) was amplified with the universal primers ITS 1 and ITS 4 (White et al., 1990). The molecular characterization was carried out by extraction of the genomic DNA by the conventional DNA extraction method with CTAB buffer. The amplified fragments were sequenced by the sequencing service of Macrogen (Seoul, Korea) using the method of Sanger et al. (1977). For P. teres, a phylogenetic analysis was performed using the ITS sequence in order to evaluate the taxonomic position of the isolates Pt M (DtLPS1) and Pt A (DtLPS2). The ingroup consisted of 30 sequences from18 species belonging to the Drechslera / Pyrenophora genera, with Brunneosphaerella jonkershoekensis used as outgroup. The sequences were aligned using the Clustal W algorithm included in the software Geneious R9 (Kearse et al., 2012). The alignment was edited automatically with the Gblock software Version 0.91b, setting the minimum length of a block at 2 and leaving all other options at default values. The best substitution model was selected using the Akaike Information Criterion (AIC) (AIC, Akaike, 1974) with the software jModelTest version 2. 1. 7 (Darriba et al., 2012) and the maximum likelihood analysis was made using the PhyML software v.3.0 (Guindon & Gascuel, 2003), using the BioNJ topology as the starting tree for the best heuristic search using nearest neighbor interchange and pruning and reinsertion of subtrees. Statistical support of the nodes was evaluated using 1000 bootstrap replicas. Sequences of both pathogen and antagonist isolates were submitted to GenBank (https://www.ncbi.nlm.nih.gov/). 2.4 In vitro antagonism assays The antagonistic abilities of Trichoderma isolates were evaluated through the dual culture technique (Dal Bello et al. 1994). Three assays were conducted with the three strains of P. teres confronted with the following 8 isolates of Trichoderma spp.: T0, T2, T3, T4, T7, T8, T9 and T10. Diameter of the colonies were measured after three and six days of incubation at 23 ºC. Percentual mycelial growth inhibition (MGI) was calculated according to the formula proposed by Michereff et al. (1994): MGI (%) = [(MGC- MGT)/ MGC] x 100, where MGC is mean length of control mycelium growth and MGT is mean length of treated mycelium growth. The experiment was randomly arrangedand each treatment had four replicates. Data taken on the sixth day were analyzed by ANOVA and Multiple Range LSD Fisher Test. The Trichoderma spp.-P. teres interaction zone was also examined under the microscope.

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2.5 In vivo evaluation of pathogenicity and plant growth-promoting parameters According to the data obtained from the in vitro tests, four Trichoderma spp. isolates (T0, T2, T3 and T9) were selected to be evaluated under greenhouse conditions. Scarlett variety barley seeds from Tres Arroyos (Buenos Aires) with 30% natural infection by P. tereswere used. The experimental design consisted of five treatments (T0/Pt A; T2/ Pt A; T3/ Pt A and T9/ Pt A and the control) and two blocks per treatment in randomized blocks (each block was a Speedling tray of 75 individual cells, with one seed per cell). The substrate consisted of 50% compost, 40% peat and 10% perlite. One of the assays was carried out at 18° C mean temperature and 70% humidity, and the other at14 ° C mean temperature and 85% humidity. To avoid any direct interaction between P. teres and Trichoderma spp. on the seed, the seeds, whose surfaces had been previously disinfected and had germinated roots, were flooded for 30 min in a solution of 1 × 108 conidia / ml of each isolate. They were drained and allowed to dry in a sterile Petri dish at 21 ± 1 ° C in darkness. After 24 hours they were sown in speedling trays. The seeds treated with the antagonist were artificially inoculated with Pt A. strain during sowing following the methodology used by Istifadah & Mc Gee (2006). A plug of active mycelium of Pt A (5×5 mm), grown for 10 days in APG at 21 ± 1 ° C, with 12 light cycles, was placed on each seed. The control seeds were immersed in sterile distilled water with 2 ml of semisolid water agar and one drop of 0.01% (v/v) Tween 20 (Biopack®, Buenos Aires, Argentina) and then inoculated with the pathogen. We evaluated parameters related to pathogenicity and plant growth promotion. Pathogenicity tests were estimated using parameter incidence and leave and stem severity. These were recorded in situ following James (1971).Severity was quantified using visual scales constructed for this purpose, one for the quantification in stems and another for leaves. Growth-promoting parameters were chlorophyll and aerial and root dry weight. Chlorophyll was estimated inSPAD units using the Minolta ® SPAD 502 chlorophyll meter. Three measurements were taken of the upper half of the second leaf of each barley seedling, and the average value was calculated. Weight of roots and stems was measured after 23 days. Forty seedlings from each treatment per block were removed. Roots were washed and dried in an oven at 60 ° C for 7 days. The aerial and radicular dry weight of each plant was recorded. 2.6 Statistical analysis The incidence data were analyzed using Mixed Generalized Linear Models (MLGM) with the InfoStat software 2014 version running on an R-DCOM dependent R platform (Baier & Neuwirth, 2007). The model proposed was a Generalized Linear Model for Binary data with fixed effects: π (x)= α + β x π represents the incidence variable of treatments (x) and β the probability change per unit of x (treatments). The link function used by the model for the binomial data family was the logit function. The means were compared by the DGC test (Di Rienzo et al. 2002). The values corresponding to the variables chlorophyll, severity of stem and leaf were analyzed by the nonparametric Friedman test. Aerial and root dry weight data were analyzed by ANOVA and Fisher LSD test. The analyses were carried out using InfoStat software, 2011version. 3. Results

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3.1 Morphocultural characterization

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Colonies of Trichoderma spp. isolates in PDA presented green coloration. The mycelium had cotton-like appearance and was floccose towards the margins except for T0, in which the conidiophores grew in pustules. All isolates except T7 and T9 released yellow pigment that spread through the agar. Isolate T0 was the only that released a distinctive odor (coconut). Sporulation ranged between 1×108 – 1×109 con/ml on PDA. All isolates of Trichoderma spp. in SNA formed pustule-like colonies. No colony released pigments to the medium and only T2, T3, T4 and T0 showed distinctive odor (coconut). In PDA medium, the growth radius of colonies of all Trichoderma isolates was more than 35 mm after 72 h at 25°C. The isolates showed growth differences at 35 °C, reaching radii between 9 and 36 mm, except T0 that reached 55 mm. On the other hand, in the SNA medium, the growth radius of Trichoderma

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colonies was greater than 20 mm after 72 h at 25°C. These also showed growth differences at 35°C, reaching radii between 8 and 24 mm, except for T0 that reached 50 mm. In microscopic observations, the branching pattern of the conidiophores was dichotomous in all isolates except for T0 in which it was irregular. The phialides of all isolates were ampuliform with almost globose conidia (L/W 1.1). Only T0 presented uncinate phialides with oblong conidia (L/W 1.1) and produced chlamydosporesIsolates A and C of P. teres showed typical cultural characteristics. The mycelium was initially grey, between dark “Mouse Grey” grey and light “Pale Mouse Grey” grey with white formations, and located close to the marginas tufts. The P. teres M isolate showed red-orange pigmentation and its mycelium was generally lighter than those of the other isolates. The conidia of the three isolates exhibited a range of colors from yellowish to olive brown, their shape was cylindrical with straight edges and rounded ends, and with two to five pseudosepta. Growth diameter measurements for the three colonies were close to 40 mm after three days of incubation in PDA, and covered the surface of the Petri dishes at six days. Conidia measurements for the three isolates were 14-19.5 × 48-65 μm.

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The tef1 sequences of Trichoderma spp. isolates were submitted to the GenBank database (Table 1). The Maximum Likelihood analysis based on the tef1, showed that T2, T3, T4, T7, T8, T9 and T10 were grouped in the same clade together with Trichoderma harzianum (AF348100 and AF348101) with a bootstrap value of 98%. On the other hand, the T0 isolate was grouped with T. longibrachiatum (DQ297069) with a bootstrap value of 99%. The maximum likelihood consensus tree is shown in Figure 1.

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The isolates P. teres M. (DtLPS1) and P. teres A (DtLPS2) were annotated in the GenBank database under accession numbers KF656728.1 and KF656729.1, respectively. The ITS sequence was grouped within the Pyrenophora teres clade with a bootstrap value of 96%. The maximum likelihood consensus tree is shown in Figure 2.

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3.2 Molecular characterization

3.3 In vitro antagonism assays

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All Trichoderma spp. isolates showed ability to reduce the mycelial growth of P. teres in dual culture in a range of 18%-54% compared to the control (Table 2). The ability to inhibit growth of the pathogen isolates showed statistically significant differences (P. teres A: F=8.53; df= 7; p = 0.0002; P. teres C: F= 5.29; df =7; p = 0.00020; P. teres M: F= 12.66; df= 7; p = 0.0001). For the three P. teres isolates, T. harzianum (T3) showed the best performance with MGI values ranging from 39% to 54% compared to the control. T. harzianum (T8) showed the lowest MGI values for P. teres A and C, although it had the second highest value for P. teres M. In general terms, the rest of the isolates showed higher MGI % for D. teres C and A than for D. teres M. A red-orange margin was observed on P. teres and in the area of contact with Trichoderma spp.; this pigmented area showed fungistatic effect against the antagonist from the second day of confrontation, which lasted two or three days depending on the Trichoderma isolate. After six days, the isolates of T. harzianum T2, T3 and T4 surrounded P. teres in areas where the pigment had not yet been produced and covered half of the colonies, sporulating on them. The isolate T. harzianum T9 surrounded them, beginning to sporulate on their margins, while T. harzianum T7, T8 and T10 began to surround them but without sporulating on them. In contrast, T. longibrachiatum T0 grew behind the pigmented area leaving a halo of inhibition in the contact area.

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The antagonistic mechanisms observed under the microscope in the zone of interaction between the three P. teres isolates and the antagonists were: mycoparasitism with coiling in the cultures with T. longibrachiatum T0 and T. harzianum T3, thin walls without characteristic pigmentation (unpigmented hyphae) with T. harzianumT2, T4 and T7, and vacuolated and deformed mycelium with T. harzianum T8, T9 and T10 (figure 3).

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3.5 In vivo evaluation of pathogenicity and plant growth-promoting parameters.

3.4 Microscopic observations

3.5.1 Pathogenicity

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All the Trichoderma isolates significantly decreased the incidence of P. teres in a range between 39% and 55% with respect to the control in both tests, with F= 14.78; p =0.0056 for assay 1 and F= 9.88; p = 0.0136 for assay 2 (Table 3). Regarding stem severity, all the isolates showed significant differences compared to the control in both assays (assay 1: T= 26.01; p = 0.0001and assay 2: T= 3.61; p = 0. 0064). These results showed a decrease ranging between 50% and 77%. For leaf severity, significant differences were found among treatments in both assays (assay 1: T= 23.27; p = 0.0001and assay 2: T= 16.30; p= 0.0001). For assay 1, T0 and T2 showed the highest severity-controlling values (60%), while for assay 2 all isolates showed high severity-controlling values, up to 70% (Table 3). 3.5.2 Plant growth promotion Regarding chlorophyll (SPAD), for assay 1 all treatments showed statistically significant differences with respect to the control (T= 7.47; p = 0.0001). The isolates T0, T2 and T9 showed the highest values, with an increase of up to 9% compared to the control. For assay 2, although the treatments did not show significant differences compared to the control (T= 1.64; p = 0.16), the highest values tended to be associated to T0, T2 and T3 (Table 4). Concerning the variables aerial and root dry weight, the treatments with highest values were different in each assay. For assay 1, regarding root dry weight, treatment with T0 showed an increase of up to 15% with respect to the control (F= 3.29; p = 0.01). For aerial weight, there were differences between treatments (F= 8.01; p = 0.0001), although T0 did not differ significantly from the control. For assay 2, no statistically significant differences were detected in dry root weight compared to the control (F= 2.11; p = 0.08), although T3 was associated with the highest values. Concerning air weight, T3 also showed an increase of up to 20% with respect to the control (F= 2.97; p = 0.02) (Table 4). 4. Discussion The correct identification of phytopathogenic agents and their antagonists is of great importance for implementing measures to prevent and manage diseases in crops of economic value. These morphocultural and phylogenetic analyses of Trichoderma isolates allowed identifying and separating two taxonomic groups: T. harzianum (T2, T3, T4, T7, T8, T9 and T10) and T. longibrachiatum (T0). Furthermore, the typical growth rate for each species is an important tool to determine the identity of new isolates. In this study, most of the T. harzianum isolates grew in accordance to the results found by Chaverri & Samuels (2003) for H. lixii/T. harzianum. On the other hand, the development of the T. longibrachiatum (T0) isolate was favored by higher temperatures, ca. 35ºC, as described by Samuels et al. (2012). With respect to the microscopical structures, T. longibrachiatum (T0) was the only isolate that showed differences in the morphology of conidiophores, phialides and conidia, supporting its assignation to a different taxonomic group (Samuels et al. 2012). While several markers such us Tef1, RNA polymerase II subunit (rpb2), calmodulin gene (cal1) and ITS have been used to elucidate the phylogenetic relationships of the Trichoderma species group (Samuels & Ismaiel 2009; Druzhinina et al. 2012), in this work the differentiation of the two taxonomic groups was possible using both phylogenetic analysis and morphological characterization. In any case, further studies including more than one molecular marker in combination with morphological, physiological and biochemical features should be performed for a comprehensive and correct identification of the T. harzianum complex. The Pyrenophora teres isolates used in this work showed colonial features similar to those found by Sivanesan (1987). These isolates presented lobulate segments in the vegetative mycelium, in agreement with Drechsler (1923); the size and shape of conidia observed in this work were also similar to those reported by Drechsler (1923) for this species. In the present work conidia catenulation was not present, in contrast with the observations recorded by Kenneth (1962).Molecular analyses of the ITS marker showed clustering of the two phenotypically different P.teres isolates within the same taxon. This confirmed its assignation to this species. Morphological and molecular components are necessary in all characterizations, because phenotypic characteristics maybe highly variable and dependent on artificial culture conditions. For in vitro interactions, the values of P. teres growth inhibition, between 18% and 54%, were similar to the results of Guigón- López et al. (2010) and Sánchez et al. (2015), who confronted Trichoderma spp. against Rizoctonia solani and Fusarium oxysporum, respectively. Likewise, the

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antagonist manifested diverse control strategies. Overgrowth and suppression of some isolates on P. teres were observed as reported by Shalini et al. (2006) and Atasanova et al. (2013) in the interaction T. harzianum- Rhizoctonia solani. In addition, competence with few signs of mycoparasitism was observed, as recorded by Atasanova et al. (2013) in the interaction between T. reesei and R. solani. A different antagonistic mechanism with T. longibrachiatum was observed. An inhibition zone, similar to the one reported by Sreenivasaprasad & Manibhushanrao (1990), suggests diffusion of non-volatile fungistatic metabolitesto the medium. Our microscopic observations of the Trichoderma-P. teres interaction zone showed mycoparasitism by coiling, in agreement with the reports of Elad et al. (1983) and Sreenivasaprasad & Manibhushanrao (1990). Another mechanism observed was the loss of turgidity of P. teres hyphae, as recorded by Sánchez et al. (2007) for the interaction T. longibrachiatum - Thielaviopsisparadoxa. The results of in vitro essays allowed selecting those isolates that showed better performances for their use in vivo assays. In these trials, both phytosanitary and growth-promoting aspects were evaluated. We agree with Bolton (2009) in that it is very important that the antagonist not only confers immunity, but also stimulates the growth of the plant, because defense mechanisms may decrease the rates of plant growth and development. With respect to phytosanitary parameters, the maximum reduction of net blotch incidence in our study was close to 60%. Similar values were found by Elad et al. (1980), Lewis & Papavizas (1985) and Inbar et al. (1994) for the incidence control of “damping-off” with T. harzianumin bean, cotton, radish and pepper. The maximum reduction of severity was in the range of 50% to 77%, similar to the values reported by Cordo et al. (2007) and Stocco et al. (2016) for the severity of Septoria leaf blotch of wheat in seeds treated with T. harzianumin a greenhouse. Furthermore, similar values were also recorded in field trials for D. tritici-repentis severity in wheat leaf with Trichoderma spp. (Perelló et al., 2003). Concerning the clorophyll variable (SPAD) an increase of up to 9% was found in plants with Trichoderma spp. compared tothe controls. Similar results were observed by Harman (2000) in maize plants with T. harzianumT-22; this author noticed that plants from treated seeds showed higher levels of SPAD than those from untreated seeds. Colla et al. (2015) also recorded higher SPAD values in lettuce, tomato and zucchini plants treated with T. atroviridae compared to non-treated ones. This suggests an invigorating effect of Trichoderma spp. In reference to the growth-promoting parameters, in our study the variables aerial and radicular dry weight of plants treated with Trichoderma spp. showed increases of up to 20% and 15%, respectively. The same effect was reported on wheat with T. harzianum, as well as greener and vigorous maize plants under Trichoderma T-22 treatments compared to untreated plants (Harman, 2000; Perelló & Dal Bello 2011). Additionally, the besttreatments for each assay differed, withT. longibrachiatum (T0) being the best for the first assay and T. harzianum (T3), for the second. These differences could be accounted for by temperature, which could have influenced the development of each of these antagonists. Temperatures close to 26°C were recorded in the first assay and, as previously mentioned, T. longibrachiatum is characterized by growing and surviving in warmer temperatures than T. harzianum (Samuels et al., 2012). Conclusions The in vitro studies carried out during this research contribute to the knowledge about the biological mechanisms involved in the Trichoderma spp.- D. teres interaction, being the first approach to this research topic in Argentina. In addition, the in vivo essays allowed assessment of the antagonistic activity of native Trichoderma spp. observed in the in vitro essays. The antagonistic isolates with best in vitro performance were also the best in vivo. Thus, these isolates may be assessed in future studies as control for other pathogens. The results of this work highlight the importance of using microorganisms adapted to the agroecological conditions of barley crops.

406 407

Acknowledgements

408 409 410 411

We are especially grateful to Dr. Pedro Balatti and Dr. Mario Emilio Ernesto Franco for his contribution to the molecular characterization of P. teres. We also thank Cecilia Morgan for providing an English review of the manuscript. This research had financial support from CONICET, CICPBA and UNLP.

412

5. References

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Table1. Geographical provenance and GenBank accession numbers of Trichoderma and P. teres isolates.

572 573 Isolates

Geographical

GenBank access

origin

number

T0

Tres Arroyos

KX572904.1

T2

Tres Arroyos

KX572905.1

T3

Bordenave

KX572906.1

T4

Tres Arroyos

KX572907.1

T7

Bolívar

KX572908.1

T8

Bordenave

KX572909.1

T9

Tres Arroyos

KX572910.1

T10

Barrow

KX572911.1

P. teres A

Los Hornos (leaf)

KF656729.1

P. teres M

Los Hornos (seed)

KF656728.1

P. teres C

Bolívar (seed)

-

574 575 576 577 578

Table 2. Mean values and standard error of percentual mycelial growth inhibition (MGI%) of P. teres A, C and M by Trichoderma longibrachiatum (T0) and T. harzianum ( T2, T3, T4, T7, T8, T9, T10).

579 Treatments

P. teres A

P. teres C

P. teres M

T0

42.59 ± 2.83 bc

45.93 ± 1.85 bc

17.78 ± 2.73 a

T2

45.56 ± 2.83 bc

41.94 ± 1.6 b

26.48 ± 2.73 b

T3

55.55 ± 2.83 d

44.45 ± 1.6 b

39.11 ± 2.12 c

T4

47.04 ± 2.83 c

45.56 ± 1.85 b

21.67 ± 2.73 ab

T7

38.52 ± 2.83 b

45.19 ± 1.85 b

35 ± 1.93

T8

27.04 ± 2.83 a

36.30 ± 1.85 a

35.93 ± 2.73 c

T9

41.85 ± 2.83 bc

51.11 ± 1.85 c

18.44 ± 2.12 a

T10

37.41 ± 2.83 b

46.30 ± 1.85 bc

27.78 ± 2.73 b

c

580 581 582 583

Values followed by the same letters do not differ significantly according to the LSD-Fisher test (P≤0.05). P. teres A (F=8.53; df= 7; p= 0.0002); P. teres C (F= 5.29; df=7; p= 0.00020); P. teres M (F= 12.66; df= 7; p= 0.0001)

584 585 586 Table 3. Variables related to pathogenicity: mean percentual values and standard error of 587 incidence and severity in stem and leaf (Assays 1 and 2). 588 Treatments

Percentage of incidence

Stem Severity (%)

Leaf Severity (%)

Assay 1

Assay 2

Assay 1

Assay 2

Assay 1

Assay 2

T0

44 ± 4 a

42 ± 5 a

12.66± 1.80 bc

10.8 ± 1.94 ab

5.9 ± 1.74

a

17.5 ± 2.9 ab

T2

35 ± 4 a

48 ± 5 a

8.59 ± 1.47 a

17.33 ± 2.85 bc

6.53 ± 1.72 ab

15.65 ± 3 a

T3

37± 4 a

42 ± 5 a

6.28 ± 0.81 a

11.45 ± 2.35 ab

9.24 ± 1.85 bc

15.65 ± 3 a

T9

47± 4 a

47 ± 5 a

13.03± 1.85 bc

8.2 ± 1.58 a

12.81 ± 2.44 d

18.4 ± 2.8 ab

Control

77± 4 b

82 ± 5 b

24.27± 1.88 e

36.5 ± 3.14 e

14.69 ± 2.57 de

51.75 ± 4.4 d

589 590 591 592 593 594

Values followed by the same letters do not differ significantly according to the DGC test (p ≤ 0.05) for incidence (Assay 1: F= 14.78; p= 0.0056. Assay 2: F= 9.88; p= 0.0136) and according to the Friedman-dms test (p ≤ 0.05) for the severity variables of stem (Assay1: T= 26.01; p= 0.0001. Assay 2: T= 3.61; p= 0. 0064) and leaf (Assay 1: T= 23.27; p= 0.0001.Assay 2: T= 16.30; p= 0.0001)

595 596 597 598 599

Table 4. Variables related to growth promotion: mean values and error standard of chlorophyll (SPAD), root and aerial dry weight (mg) (Assays 1and 2).

600 Treatments

Chlorophyll (SPAD)

Dry root weight (mg)

Dry air weight (mg)

Assay 1

Assay 2

Assay 1

Assay 2

Assay 1

Assay 2

T2

29.83 ± 0.3 bc

32 ± 0.39 a

34.7 ± 1.2 a

12.5± 1 ab

45.1 ± 1.6 ab

28.4±1.3 ab

T3

28.85 ± 0.29 b

31.94 ± 0.37 a

34.2 ± 1.5 a

14.2± 1.2 b

49.7 ± 2 bc

32.3±1.6 b

T9

29.26 ± 0.76 bc

31.12± 0.30 a

32.9 ± 1.7 a

12.5± 1.2 ab

42.1 ± 1.5 a

25.4±1.5 a

T0

29.86 ± 0.51 c

31.97± 0.38 a

39 ± 1.3

b

9.7± 0.79 a

53.1 ± 1.5 c

26.6±1.3 a

Testigo

27.44 ± 0.51 a

31.33± 0.30 a

33.9 ± 1.2 a

12.7±1.3 ab

52.8 ± 2

26.7±2.2 a

c

601 602 603 604 605 606 607 608 609 610 611

Values followed by the same letters do not differ significantly according to the LSD-Fisher test (P≤0.05) for the root dry weight (Assay 1: F= 3.29; p= 0.01. Assay 2: F= 2.11; p= 0.08) and shoot dry weight variables (Assay 1: F= 8.01; p= 0.0001. Assay 2: F= 2.97; p= 0.02) and according to the Friedman-dms test (p ≤ 0.05) (Assay 1: T= 7.47; p = 0.0001. Assay 2: T= 1.64; p= 0.16) for chlorophyll.

612 613 614 615 616 617 618 619 620 621 622 623 624 625

Highlights  Trichoderma spp. exerts mycoparasitism and antibiosis against Pyrenophora teres in vitro studies.  New argentine isolates of Trichoderma spp. exert biological control against net blotch of barley.  Trichoderma harzianum (T3) and T. longibrachiatum (T0) increase the aerial and radicular dry weight and the chlorophyll (SPAD) of plants treated.  In Argentina the study of interaction between Trichoderma spp and the pathosystem Pyrenophora teres- barley is a novel approach.

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CRediT author statement

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Paulina Moya: Conceptualization, Methodology: Conducted in vitro and in vivo assays, performed molecular characterization and statistical analyses, Writing- original draft preparation. Viviana Barrera: Methodology: performed molecular characterization and phylogenetic analyses. Josefina Cipollone, Carolina Bedoya and Lucila Kohan: Methodology: Contributed with in vivo assays. Marina Sisterna and Andrea Toledo: Supervision, Validation, Writing-Reviewing and Editing, Secured funding. All authors read and approved the manuscript.

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